The use of recombinant adenoviral vectors for delivery of genes or combinations of genes to tumor cells has been widely investigated. (Shewach et al., 1994, Davidson, 1993, Ross et at., 1995, Boviatsis et al., 1994, Barba et al., 1994, Wei et al., 1994, Mansour et al., 1985, Smythe et al., 1995, Goebel et al., 1995, Perez-Cruet et al., 1994, Chen et al., 1994, and flung et al., 1988). Recombinant adenoviruses are easily engineered to express the gene or genes desired for direct cell killing, for induction of host immune responsiveness to tumor cells, or both, and do not require mitotic activity for gene transfer expression (Davidson et al., 1993, Davidson et al., 1994, and Akli et al., 1993). Further, recombinant adenoviruses can be amplified to high titers and are amenable to formulation (Mulligan, 1993).
The ability to purify recombinant adenovirus to high titers is critical given the inefficiency of gene transfer to elements such as gliomas with a vector system. In vitro, efficacy is not noted with multiplicities of infection (MOI) below 100 infectious units per cell (Shewach et al., 1994, Chen et al., 1994). It is suboptimal below 250-500. The need for high titers, coupled to the fact that only one to five percent of virions in purified viral preps are infectious, requires high particle titers in the dosing inoculum. The resulting inflammatory response in the dosing inoculum limits the duration of gene transfer and subsequent redosing regimens (McCoy et al., 1995, McCoy et al., 1995, Barr et al., 1995, and Brynes et al., 1995).
Another problem often encountered in gene therapy regimens has to do with the host immune response following gene vector delivery. The host immune response following adenovirus delivery is robust in all tissues including brain (references). In tumor bearing hosts the response may be beneficial for therapy but detrimental to subsequent vector delivery. The T-cell response to virally infected cells may enhance killing of tumor cells (ref), but B-cell activation may result in the production of a neutralizing antibody response which limits re-infection (ref). Redosing must therefore be accomplished following an initial infection with lower titers (often not efficacious) or with virus formulated to reduce its antigenicity.
Two aims to improve the effectiveness of adenoviral mediated gene transfer for glioma therapy are to increase the efficiency of gene transfer and reduce the need for frequent re-dosing regimens. The latter can be accomplished by the sustained release of low dose adenovirus from biodegradable microspheres into the tumor mass.
Biodegradable microspheres have been successfully used to deliver drugs at a controlled rate to specific tissues, such as brain (Cohen et al., 1991, Gref et al., 1994, Okada et al., 1991, Pekarek et al, 1994, Sanders et al., 1986, Sabel et al., 1990, Judy et al., 1995, Brem et al, 1994, and Walter et al., 1994). The long term goal for the encapsulation of viral vectors for gene therapy for such diseases as tumors of the supportive glia of the brain is to provide sustained local release. As such, formulations must contain concentrated viable virus in acceptable volumes for delivery and the tissue reaction to the polymer must be minimal.
Pharmaceutical research has led to the identification of many reagents compatible with controlled delivery of drugs enterically and systemically. These reagents include hydrogels, self-diffusion and self-regulated systems, microparticles, biodegradable polymers and porous membranes (Pitt, 1990, Eldridge et al., 1991, and Eldridge, 1989). Hydrogel systems were first used for the delivery of insulin in diabetic rat models (Davis, 1972) and provide an aqueous microenvironment for the diffusional migration of macromolecular compounds. These gels limit the migration of macromolecules with a release dependent upon the polymer content of the gel and the molecular weight of the encapsulated substrate (Jhon and Andrade, 1973. Zentner et al., 1979). Self-diffusion systems allow for sustained release that it is dependent upon hydration (Sabel, 1990, Langer and Folkman, 1976, Langer and Folkman, 1978, and Langer et al., 1980), while self-regulated systems allow for controlled release by an effector molecule, (Brownlee and Cerami, 1979, Brownlee and Cerami, 1983).
Poly(lactide-co-glycolide) (PLG) copolymers have been well-characterized an offer many advantages for the sustained release of macromolecular preparations. First, PLG has well established biocompatibility and has been shown to be safe in in vivo settings (Redding et al., 1984). Degradation of PLGA in vivo occurs by acid or base catalyzed hydrolysis (Mason et al., 1981) and results in production of lactic and glycolic acids with minimal inflammatory responses (Visscher et al., 987, Tice and Cowsar, 1984). Importantly, hydrolysis rates can be adjusted by modifying the monomer ratios of the glycolic and lactic acid components (Miller et al., 1977).
Several prior art patents relate to the microencapsulation of biologicals. For example, U.S. Pat. No. 4,948,586 to Bohm et al., issued Aug. 14, 1990, discloses a microencapsulated insecticidal pathogen for application to vegetation. The insecticidal viral, bacterial, or fungal pathogen is encapsulated in a polymeric encapsulating agent.
U.S. Pat. No. 5,463,092 to Hostetler et al., issued Oct. 31, 1995, discloses lipid containing prodrugs for treating viral infections. The compounds comprise phosphonoacids having anti-viral activity which are linked to one of a selective group of lipids. A liposome is formed, at least in part, from the compositions disclosed.
U.S. Pat. No. 5,160.745 to DeLuca et al., issued Nov. 3,1992, discloses a microencapsulated biologically active macromolecular agent. The microencapsulant is a biodegradable vinyl derivative.
Referring specifically to glioblastoma, the development of alternative forms of treatment for glioblastoma, such as gene therapy, is warranted and the success of which is becoming more predictable (Moolten and Wells, 1990, Ezzeddine et al., 1991, Culver et al., 1992, Oldfield et al., 1993, Ram et al., 1993, Ram et al., 1992, Short et al., 1990, and Takamiya et al., 1993). The first clinical trials using gene therapy for the treatment of glioblastoma multiform used in situ implantation of murine retroviral producer cells. The gene transferred expressed Herpes simplex type I (HSV-1) thyrnfidine kinase (tk). This herpes gene product is not toxic but has specificity for the antiviral 2'-deoxyguanosine analog pro-drug, ganciclovir. Once phosphorylated, this drug is highly toxic to mitotically active cells (Shewach et al., 1994). Because the murine based retroviruses infect only dividing cells (Moolten and Wells, 1990, Ezzeddine et al., 1991, Culver et al., 1992, and Ram et al., 1993) and in humans with glioblastoma multiform, only a small percentage of tumor cells are dividing, re-implantation of murine cells and additional rounds of high dose ganciclovir therapy are required. Immune responses to xenografts and systemic toxicity to ganciclovir are limitations of this approach.
Inflammatory bowel disease (IBD) is a second disease for which gene transfer studies have been initiated using recombinant viral vectors as a first step towards the development of an alternative therapy for the disease. IBD is estimated to affect 600,000 Americans with 40,000 new cases per year. Disabling symptoms of this disease usually begin in adolescence or early adulthood (Peppercorn Mass. 1992). Currently employed therapies directed at limiting chronic inflammation in IBD include drugs such as corticosteroids, 5-aminosalicylate (5-ASA) and immunosuppressives (e.g. irniran and 6-mercaptopurine) that have wide ranging affects on the immune system and serious side effect profiles (Peppercorn Mass. 1992).
Current understanding of the development and perpetuation of chronic intestinal inflammation in IBD suggests that there is a balance between pro-inflammatory and anti inflammatory factors. There is significant redundancy in the system so that multiple factors have overlapping roles; clearly it is the combined interaction of multiple factors that results in intestinal inflammation. Interleukin 1 (IL-1) is fundamental to many inflammatory processes including IBD (Blumberg et al., 1995, Dinarello et al., 1993, Isaacs et al, 1992). Studies suggest that it is consistently up-regulated in inflamed tissue in IBD and in many animal models of IBD (Kunkel et al., 1991. Isaacs et al, 1992). Studies of inflamed tissue from IBD patients suggest that there is an imbalance between the production of the proinflammatory IL-1 and its naturally occurring antagonist interleukcin 1 receptor antagonist protein (IL-1ra) favoring the production of IL-1 (Isaacs et al, 1992). An overall goal in the treatment of IBD would thus be to increase local expression on IL-1ra in an attempt to counterbalance the proinflammatory effects of IL-1.
An increased understanding of cytokine networks and the mechanisms of paracrine interactions between epithelial cells, mesenchymal cells, neurons and inflammatory cells in the intestinal tract has led to the development of several candidate targets for therapy of IBD. In addition to IL-1ra, anti-TNF-.alpha. monoclonal antibody and interleukin 10 (IL-10) represent cytokine-related putative therapeutic agents targets that are currently in human trial (Dullemen et al, 1995.). Delivery of these agents to the sites of active inflammation has been problematic. Systemic administration is poorly tolerated for chronic diseases such as IBD and it is difficult to deliver adequate local concentrations of the agent to perform paracrine and autocrine type functions. Since most of these putative therapeutic agents are proteins oral delivery remains difficult.
Numerous animal models for IBD, such as the rabbit model of formalin-immune complex colitus (Comanelli et al., 1990.), IL-10 knockout mice (IL-10T mice) (Kuhn, et al., 1993), IL-2 deficient mice (Sadlack et al., 1993), and the T-cell receptor knockout mouse (Mombaerts et al., 1993), enable the determination of whether cytokine-mediated therapy can inhibit the onset and progression of disease. Treatment of rabbits having formalin-immune complex colitus with bolus i.v. injections of IL-1ra protein significantly reduced the inflammatory response associated with the experimental colitus (Cominelli et al., 1990). Further, in IL-10T mice, bolus injections of IL-10 (10 ug) 30 minutes prior to challenge with lipopolysaccharide (LPS), prevented endotoxic shock. More importantly, treatment of IL-10T mice by i.v. injection of an adenovirus containing the human IL-10 cDNA protected 67% of animals (n=6) from LPS challenge 9 days after infection. Those animals that survived the challenge were found to contain high plasma levels of IL-10 (&gt;200 picograms/ml) and all of these animals withstood a second challenge 20 days after adenoviral infection. Animals (n=7) that were infected with Ad.RSVlacZ at similar doses did not survive LPS challenge. It can be concluded from these studies that adenoviral-mediated expression of pharmacologically relevant levels of gene products can alter the natural history of diseases such as IBD.
It is desirable to provide a delivery system for functional gene vectors and specifically for recombinant adenovirus. It is further desirable to derive a biodegradable microsphere used for delivery system deliver functional genes. More specifically, it would be desirable to provide a means for delivering adenoviral vectors for gene therapy such as for the treatment of intracerebral glioma, inflammatory bowel disease, and other diseases.